Bidirectional promoters are abundant in the genome. They are defined as a shared regulatory region that falls in the intergenic space between two oppositely oriented genes that are separated by no more than 1,000 bp. These genes are organized in a head-to-head arrangement and transcribed away from one another. The closely spaced arrangement of the transcription start sites (TSSs) suggests that each pair of genes is recognized as a nonrandom event in the genome, proven by the fact that a greater than expected number of promoters have this architecture. One plausible scenario for the creation of bidirectional gene pairs is chromosomal recombination bringing the ends of two TSSs in close proximity. This union is likely to be irreversible because breakage of the intergenic promoter region would interrupt the normal regulation of two genes, profoundly affecting normal genomic function, such as DNA repair. We have mapped bidirectional promoters in numerous vertebrate genomes. In doing so, we are creating the first high-confidence regulatory map of bidirectional promoter sequences shared in vertebrate lineages as well as identifying transcripts that are unique to single vertebrate lineages, including primates. We have identified the first examples of lineage-specific transcripts in human and in cow using this approach. (Piontkivska H, Yang MQ, Larkin DM, Lewin HA, Reecy J, Elnitski L , BMC Genomics 2009 and Bovine Genome Consortium, including Elnitski L, Science 2009). In additon to bidirectional promoters we are investigating the specific activation of alternative promoters of genes and finding that their diverse and varied patterns have implications for understanding the etiology of diseases as well as the use of gene therapies (Jacox E, Gotea V, Ovcharenko I, Elnitski L PLoS One 2010). In addition to characterizing promoter regions, we are interested in identifying novel types of elements such as negative regulators of gene expression (NREs;Petrykowska H, Vockley C, and Elnitski L, Genome Research 2008). In contrast to the large body of literature on positively acting elements such as enhancers and promoters, cis-acting NREs have not been extensively studied. Despite their scarceness in the literature, these elements are likely to be abundant in the genome. Examples of NREs include silencers, which decrease expression of a gene under their regulation and enhancer-blocking (EB) elements, which prevent the action of an enhancer on a promoter when placed between the two, but not otherwise. By developing a strategy to experimentally test NREs, we have identified novel examples of these functions in the human genome. This research provides the opportunity to test regions of the genome whose function is not known. Furthermore, as functional elements in the genome, silencers and enhancer-blockers are likely to be targets of mutations involved in human diseases. (ENCODE Consortium, PLoS Biol 2010). Exonic splicing enhancers (ESEs) are a third category of regulatory elements affecting gene expression. We have compared all former predictive methods for ESEs to show that some approaches are more precise than others. We have produced an online toolkit to test polymorphisms that occur in coding sequences to assess whether they may affect mRNA splicing (http://research.nhgri.nih.gov/skippy). In addition to the web-based server, stand-alone software is available to the community for download. This software allows researchers to screen an unlimited number of variants of unknown function computationally, such as might be generated in whole exome sequencing analyses. (Woolfe A, Mullikin JC, and Elnitski L, Genome Biology 2010). All of these research projects examine cis-acting elements and converge on the identification of transcription factor binding sites or splicing regulators, which are bound by trans-acting proteins and represent the basic components of gene regulation. Regulatory motifs have been published for each of the projects above. During the course of our work, novel motifs were implicated as silencers and new biological insights were revealed for the regulation of alternative promoters. A collaborative effort has been established to define the regulatory networks involving these motifs, especially in biological pathways implicated in cancer (Lichtenberg J, Kurz K, Liang X, Al-ouran R, Neiman L, Nau LJ, Welch JD, Jacox E, Bitterman T, Ecker K, Elnitski L, Drews F, Lee SS, Welch LR BMC Bioinformatics 2010). Moreover, we are building tools to assess epigenetic events that aberrantly affect gene expression in tumors versus normal cells. Using an Illumina Infinium methylation array platform we have investigated the DNA methylation pattern in 36 ovarian tumors and an additional 12 normal samples. These are being compared to methylation patterns in endometrial tumors and metastatic tumors from the uterus to the ovary. In conjunction with DNA methylation, RNA-seq was performed on a large number of tumor and normal samples. The results identify loci with altered DNA methylation patterns whose transcript levels have also changed from the normal pattern. We are addressing the presence of specific mutations in these tumors which might connect the gene expression and DNA methylation patterns to a testable feature.